This invention relates to a novel uniform solid-phase composition which makes possible simple and rapid determination of .beta.-hydroxybutyric acid and to a method for preparing the said composition.
.beta.-Hydroxybutyric acid, a kind of ketone body, is an intermediate product of fatty acid metabolism and is found in large quantities in the body fluids (blood, urine, etc.) of diabetes patients and diabetes animals. The invention is hereinafter described in detail on the case of determining .beta.-hydroxybutyric acid in body fluids.
A carbohydrate metabolic disorder such as insulin deficiency due to pancreas incretion disorder causes fatty acid oxidation in the liver to increase. This results in abnormally increased ketone bodies such as acetone, acetoacetic acid, and .beta.-hydroxybutyric acid, which are excessively accumulated in tissues, blood, and urine (ketosis state). It is necessary, therefore, in the case of high-degree pancreas incretion disorder, particularly serious diabetes, or when dosing a patient with carbohydrate limiting food, to pay attention to the ups and downs of the ketone bodies in the body fluid.
It is generally said that when ketosis state occurs in series diabetes, .beta.-hydroxybutyric acid of these ketone bodies in the blood remarkably increases, resulting in increased (.beta.-hydroxybutyric acid)/(acetone+acetoacetic acid) ratio, and thus most sharply indicates abnormal metabolism.
A known quantitative determination method of .beta.-hydroxybutyric acid is the method by Williamson et al which uses a .beta.-hydroxybutyric acid dehydrogenase (Biochemical Journal, Vol. 82, p. 90, 1962). In this method, .beta.-hydroxybutyric acid is reacted with .beta.-hydroxybutyric acid dehydrogenase and diphosphopyridine nucleotide (DPN), and the absorbance of the produced diphosphopyridine nucleotide of reduced type (DPNH) is measured at the specific absorption of 340 nm. The quantity of .beta.-hydroxybutyric acid is determined from the molecular extinction coefficient. Accordingly, there are problems that a high-class ultraviolet spectrophotometer is required, and high skill and long period of time are needed in analysis.
To eliminate such drawbacks, there has been developed a simple colorimetry of high-sensitivity of ketone body in blood, which requires no ultraviolet spectrophotometer (Tokkai Sho 55-035232). In this method, .beta.-hydroxybutyric acid is first acted with .beta.-hydroxybutyric acid dehydrogenase and nicotineamideadenine dinucleotide (NAD) to be oxidized enzymatically into acetoacetic acid, and then reacted with p-nitrophenyldiazonium fluoroborate into an azo compound, together with the acetoacetic acid contained originally in the blood. The azo compound is reduced with hypophosphorous acid into a stable hydroazo compound, and the total acetoacetic acid is determined by colorimetry of the hydroazo compound by use of the wave length of 390 nm. Separately, only the acetoacetic acid originally contained in the blood is measured by similar colorimetry but without action of .beta.-hydroxybutyric acid dehydrogenase. The above total quantity of acetoacetic acid is subtracted by this acetoacetic acid quantity to give the quantity of .beta.-hydroxybutyric acid in the blood. This method, however, has still had a number of problems as a daily clinical examination method, requiring high skill and long time for measurement because of pretreatment (protein removal) of the blood sample and complex operation, and needing special utensils and a high-class spectrophotometer.
As the simpler examination measure, there is Owen's tester for determining .beta.-hydroxybutyric acid (U.S. Pat. No. 4,351,899). Owen's tester has two absorptive test surfaces wherein the first absorptive test surface contains tetrazolium salt, NAD, and electron transporter, and dried residue of .beta.-hydroxybutyric acid dehydrogenase, and the second absorptive test surface contains dried residue of pH buffer. In using the test indicator, the first test surface is put on the second test surface, and a material is dropped on them, and the concentration of .beta.-hydroxybutyric acid in the material can be measured by reading the hue after 2 min from dropping. It maintains the stability of the test indicator that it has two absorptive test surfaces. Owen states that if tetrazolium salt, NAD, an electron carrier, .beta.-hydroxybutyric acid dehydrogenase, and pH buffer for maintaining pH in alkaline side, which are reagents necessary for determining .beta.-hydroxybutyric acid, are dissolved in one solution, color development occurs.
Owen's test indicator has surely made a progress in simplicity. However, it is troublesome that a sample has to be dropped on the two absorptive test surfaces put on each other after taken out from separate vessels for drying. Further, although both reagents on the first and second absorptive test surface have to be dissolved uniformly to advance the reaction quantitatively, only dropping a sample on the two absorptive surfaces put on each other can not provide a uniform reaction condition. Accordingly, Owen's test indicator is lacking in quantitativity as a major problem.